Download Wasp Voodoo Rituals, Venom-Cocktails, and the Zombification of Cockroach Hosts SYMPOSIUM Frederic Libersat

Integrative and Comparative Biology Advance Access published April 4, 2014
Integrative and Comparative Biology
Integrative and Comparative Biology, pp. 1–14
doi:10.1093/icb/icu006
Society for Integrative and Comparative Biology
SYMPOSIUM
Wasp Voodoo Rituals, Venom-Cocktails, and the Zombification
of Cockroach Hosts
Frederic Libersat1 and Ram Gal
Department of Life Sciences, Ben-Gurion University of the Negev, P.O. Box 653, Be’er Sheva 84105, Israel
1
E-mail: [email protected]
Synopsis The parasitoid Jewel Wasp uses cockroaches as a live food supply for its developing larvae. The adult wasp uses
mechanoreceptors on its stinger to locate the host’s cerebral ganglia and injects venom directly into the cockroach’s
‘‘brain,’’ namely in the subesophageal ganglion and in and around the central complex in the supraesophageal ganglion.
As a result, the cockroach first engages in continuous grooming for roughly 30 min. Dopamine identified in the wasp’s
venom is likely to cause this grooming, as injecting a dopamine-receptor antagonist into the cockroach hemolymph prior
to a wasp’s sting greatly reduced the venom-induced, excessive grooming. Conversely, injecting a dopamine-receptor
agonist into the brain induces excessive grooming in normal cockroaches. A second effect of the head-sting is the
induction of a long-lasting lethargic state, during which the cockroach demonstrates a dramatically reduced drive to
self-initiate locomotion. Unlike most paralyzing venoms, Ampulex’s venom seems to affect the ‘‘motivation’’ of its host to
initiate locomotion, rather than affecting the motor centers directly. In fact, the venom specifically increases thresholds
for the initiation of walking-related behaviors and, once such behaviors are initiated, affects their maintenance without
affecting the walking-pattern generators. Thus, the venom manipulates neuronal centers within the cerebral ganglia that
are specifically involved in the initiation and maintenance of walking. We have shown that in stung cockroaches focal
injection of an octopaminergic receptor agonist around the central complex area in the brain partially restores walking.
Another likely candidate target of the venom is the opioid system, which is known to affect responsiveness to stimuli in
insects. Opioid receptor agonists increase startle threshold in control cockroaches and using a bioassay for opioid
receptors, we found that the venom blocks opioid-like receptors. This effect is reversed with naloxone, an opioid
antagonist.
Introduction
Many parasitoid wasps use insects or spiders as a
food supply for their developing larvae (Quicke
1997; O’Neill 2001). To this end, the adult female
typically stings the host, penetrating its cuticle with
the ovipositor and injecting a venom cocktail into
the hemolymph. In most cases the venom blocks
the neuromuscular junction and paralyzes the host.
The wasp then lays one or more eggs on, or inside,
the body of the host, then conceals the host in a nest
or a burrow for protection (for review, see Libersat
and Gal 2007). Some parasitoid wasp species, however, exert a more sophisticated modulation of their
host. These wasps may affect the central nervous
system (CNS) of their hosts and induce or repress
specific behaviors that accommodate the developmental constraints of their larvae (Libersat et al.
2009). For example, the braconid endoparasitoid
wasp Glyptapanteles coerces its caterpillar host to behave as a bodyguard to protect its larvae (Grosman
et al. 2008), and the larva of the Ichneumonid wasp
Hymenoepimecis manipulates its spider host to stop
building its normal orb-shaped web and to begin
building a shelter-like web that will protect the
larvae during metamorphosis (Eberhard 2000).
These, and other, examples (Libersat and Gal 2007)
provide unique insights not only into the evolutionary processes involved in the establishment of such
exquisite predator–prey or parasite–host adaptations,
but also into the mechanisms underlying both the
ß The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved.
For permissions please email: [email protected].
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
From the symposium ‘‘Parasitic Manipulation of Host Phenotype, or How to Make a Zombie’’ presented at the annual
meeting of the Society for Integrative and Comparative Biology, January 3–7, 2014 at Austin, Texas.
2
feed on nutritious hemolymph for the next few days.
When ready to pupate, the larva penetrates through
the cockroach’s cuticle, feeds on its internal organs
and pupates inside the abdominal cavity. There, well
satiated and safe from predators, the larva metamorphoses and emerges roughly a month later as an
adult, ready to restart this unique life cycle.
The second sting into the head thus evokes two
different and specific behavioral manipulations in
the cockroach prey: a relatively short phase of excessive grooming and a significantly longer inhibition of
the cockroach’s escape behavior and spontaneous
locomotion. These behavioral observations raise a
number of interesting questions: does the wasp
inject its venom directly into the CNS of its host?
If so, to which CNS region(s) is the venom injected
and how does the wasp identify the target? What
neuronal modulations account for the excessive
grooming behavior, and which circuits are affected,
and how, to induce the long-lasting decrease in
escape responses and locomotion? These are the
main questions that we intend to answer in this
review.
The head-stinging process
The site of the Jewel Wasp’s sting, as well as the
unique effects of wasp’s venom on the behavior of
its cockroach host, suggest that the wasp uses its
stinger—a modified ovipositor—to inject venom directly inside the head ganglia of its host. However,
until 2003, a direct proof of such injection of venom
into the host’s CNS was lacking. Furthermore, it is
only recently that the sensory mechanisms allowing
the wasp to inflict this ‘‘stereotactic’’ sting have been
identified.
The Jewel Wasp injects venom directly inside the
cockroach’s CNS
To test the specific location of injection, Haspel et al.
(2003) injected Jewel Wasps with a mixture of C14
radiolabeled amino acids that were incorporated into
the venom. In cockroaches stung by such ‘‘hot’’
wasps, most of the radioactive signal was localized
around the midline of the sub-esophageal ganglion
(SEG) and in the central part of the supra-esophageal
ganglion (SupEG), posterior to the central complex
(CC) and around the mushroom bodies (Fig. 2A).
This means that the wasp’s stinger penetrates
through the protective ganglionic sheath of these
two head ganglia (the cockroach’s ‘‘brain’’), such
that venom is injected directly onto the neuronal
targets. However, how can such a precise venom injection be achieved?
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
wasp’s and its host’s behavior. Unfortunately, however, the mechanisms used by most behavior-manipulating parasitoid wasps, namely the chemicals
involved, their means of delivery, their neuronal targets, and their methods of action within the host’s
CNS, are today little understood.
One exception to this and probably the best and
only well-understood example of underlying neuronal mechanisms of a wasp-induced behavioral manipulation is the relationship between the ampulicine
Jewel Wasp (Ampulex compressa Fabricius) and its
cockroach host (Libersat 2003) (Fig. 1). To supply
its larvae with live cockroaches (Periplaneta americana) as a food supply, this tropical wasp first attacks
a cockroach by clamping its mandibles on the pronotum or on the base of the wing and inflicting a
sting into the host’s thorax. This sting temporarily
inactivates central motor circuits to block motor
output in the cockroach’s forelegs for 1–2 min
(Moore et al. 2006), facilitating a second and
longer sting, through the host’s neck and into its
head. Then, after the head-sting is complete, the
wasp leaves for roughly 30 min in search of a nest
suitable for the development of its young; during this
time, the stung cockroach shows a peculiar behavioral change: instead of escaping the scene, it engages
in excessive grooming behavior and thoroughly
cleans its entire body with its legs and mandibles
(Weisel-Eichler et al. 1999). However, it is only
when the wasp returns to the grooming cockroach
that the full and most exquisite extent of the stinginduced behavioral modulation can be observed. At
this stage, between 30 and 40 min after the sting and
lasting a few days, the cockroach becomes what can
be figuratively described as a docile ‘‘zombie’’ that is
completely compliant with the wasp’s needs. Upon
returning to its stung prey the wasp first uses its
strong jaws to break the cockroach’s antennae close
to their base and then sucks up the exuding fresh
hemolymph from the stumps. The stung cockroach,
nevertheless, neither flees nor fights off the wasp,
allowing its predator to grab one of its antennal
stumps and escort it in a long walk to the preselected nest. Guided by the wasp, the docile cockroach enters its ‘‘tomb’’ and remains immobile as the
wasp lays an egg and glues it onto the mid-leg cuticle
(Fouad et al. 1994). Although not paralyzed, the
‘‘zombie’’ cockroach does not escape the nest as
the wasp searches around for small items (leaves,
pebbles etc.) with which to seal the entrance,
before flying away. Entombed inside the sealed
nest, the stung cockroach then serves as living food
storage for the developing larva that hatches a couple
of days later and perforates the host’s leg cuticle to
F. Libersat and R. Gal
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
Fig. 1 Life cycle of the parasitoid Jewel Wasp and the effects of its venom on cockroach behavior Upon striking the cockroach, the female Jewel Wasp grabs it by the pronotum and stings it in
the thorax. This sting induces a short-term (3–5 min) flaccid paralysis of the front legs, which is mediated by venom compounds that open chloride channels in thoracic neurons. This paralysis
facilitates a second, and longer, sting in the head, after which the wasp grooms thoroughly, leaves the cockroach and goes in search for a suitable nest (here, a glass vial). In the meantime, as a
result of the head-sting, the cockroach grooms excessively for 30 min. This is presumably due to venom-induced activation of dopamine-related circuits within the cockroach’s brain. The wasp
then returns to the cockroach, cuts off both its antennae and drinks the hemolymph oozing from the stumps. At this stage, the cockroach enters the third phase of the envenomation process,
namely a ‘‘lethargic’’ state during which it does not self-initiate walking or escape responses. The wasp then transports the cockroach to the nest, holding its antennal stump and walking
backwards while the ‘‘lethargic’’ cockroach follows in a docile manner. Next, the wasp glues one egg onto the cockroach’s leg, seals the nest with leaves and pebbles, and then leaves. Under
laboratory conditions, the cockroach will remain ‘‘docile’’ for 3–6 days and then resume normal activity. However, in the natural setting, the wasp’s larva hatches from its egg and feeds of the
cockroach’s hemolymph. After a few days, it penetrates the host’s abdomen to feed on its internal organs, ultimately killing the cockroach before the venom’s effect dissipates. The larva then
pupates inside the cockroach’s abdomen, metamorphoses and hatches as an adult approximately a month later.
Wasp ‘zombifies’ cockroach host
3
4
F. Libersat and R. Gal
The Jewel Wasp uses sensory cues from its stinger
to identify the cockroach’s CNS
In a recent study, Gal et al. (2014) hypothesized that
specialized sensory organs on the wasp’s stinger
(Fig. 2B) allow the wasp to discriminate the host’s
brain from other tissues inside the head capsule. In
this scenario, the wasp would identify the brain in a
manner similar to the mechanism that other parasitic
wasps use to locate a hidden host within a surrounding substrate (LeRalec et al. 1996; Vilhelmsen 2000;
Vilhelmsen et al. 2001). To test this hypothesis,
possible sensory organs on the stinger were ablated
with liquid nitrogen and the wasps were allowed to
sting normal cockroaches. The treatment prolonged
the stinging duration more than 20-fold, suggesting
that sensory ablation opened a sensory feedback
loop during the stinging process. Next, using a complementary approach, the ultimate target of the stinger, namely the SupEG, was completely removed
from the head capsule of cockroaches prior to the
wasp’s sting. Indeed, as shown in Fig. 2C, this treatment significantly prolonged the duration of the
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
Fig. 2 The parasitoid Jewel Wasp uses mechanoreceptors to identify the brain of its cockroach host (A) The SupEG (top) and SEG
(bottom) of a cockroach stung by a radiolabeled wasp (autoradiographs). Black staining indicates the presence of venom in and around
the central complex of the SupEG and throughout the entire SEG. Adapted from Haspel et al. (2003). (B) The tip of the wasp’s stinger
(scanning electron micrograph). Each of the three valves that comprise the stinger’s shaft possesses specialized sensilla (arrows).
Adapted from Gal et al. (2014). (C) In a set of behavioral experiments, cockroach brains were either removed (No Brain) or replaced
with soft or hard agarose pellets prior to a wasp’s sting. Whereas the duration of the head sting was significantly prolonged in ‘‘No
Brain’’ and ‘‘Soft Agarose’’ cockroaches compared with ‘‘Control’’ cockroaches, it was not changed in ‘‘Hard Agarose’’ cockroaches. In
addition, wasps injected venom inside Hard Agarose pellets but not inside Soft Agarose pellets. The duration of the wasp’s first sting in
the cockroach’s thorax (wherein no neuronal tissue was manipulated) remained unchanged in all groups. Bars represent means SD,
***P50.001 (Kruskal–Wallis ANOVA). Adapted from Gal et al. (2014).
Wasp ‘zombifies’ cockroach host
Venom-induced grooming behavior
and the involvement of dopamine
Following recovery from the transient paralysis of the
front legs, cockroaches stung by the Jewel Wasp
groom almost continuously for 30 min (Fig. 1).
The sting-evoked grooming is a complex behavior
involving coordinated movements of different appendages (Fig. 3A) and exhibits all the components
of normal grooming behavior. It occurs only when
the wasp inflicts the head-sting and cannot be accounted for by the stress of the attack, contact
with the wasp, or mechanical irritation (Fig. 3B),
suggesting that the venom recruits central neural networks responsible for grooming (Weisel-Eichler et al.
1999).
Studies of the sting-induced grooming suggest that
monoaminergic systems within the cockroach’s CNS,
and most prominently dopaminergic systems, are
involved in activating this excessive grooming. For
instance, an excessive grooming behavior that resembles the sting-induced grooming can be induced in
un-stung cockroaches by injecting the monoaminereleasing drug reserpine into the SEG, by injecting
dopamine (DA) into the hemolymph (Fig. 3B) or by
injecting a D1 DA-receptor agonist into the SupEG
(Fig. 3C) (Weisel-Eichler et al. 1999; Weisel-Eichler
and Libersat 2002). On the other hand, injecting a
DA-receptor antagonist [but not octopamine (OA)receptor antagonists] into the cockroach hemolymph
prior to a wasp’s sting greatly reduced the venominduced excessive grooming (Weisel-Eichler et al.
1999) (Fig. 3D). Finally, injecting a DA-receptor agonist into the SupEG of stung cockroaches failed to
evoke excessive grooming, suggesting a venominduced change in the DA-mediated signal-transduction cascade at, or downstream of, the DA-receptors
(Weisel_Eichler and Libersat 2002). A DA-like substance identified in the wasp’s venom (Weisel-Eichler
et al. 1999) is likely to be the component that induces this excessive grooming. Interestingly, D1 DAreceptor agonists have been shown to increase grooming behavior in the fruit fly Drosophila melanogaster
(Yellman et al. 1997) as well as in mammals (Molloy
and Waddington 1984), possibly indicating shared
analogous mechanisms for this complex and coordinated behavior in evolutionary-distant organisms.
A grooming-inducing venom cocktail has been reported only in the Jewel Wasp. However, is there an
evolutionary advantage for the wasp in inducing this
excessive grooming behavior, or is it simply a consequence, or a side-effect, of the venom injected into
the head ganglia? Although this is obviously a hard
question to answer, some alternative (but not necessarily mutually exclusive) possibilities come to mind.
First, considering the wasp’s life cycle, a crucial function of the sting is the production of a long-lasting
hypokinetic effect in the cockroach. For reasons yet
to be determined, this effect commences only within
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
head-sting, indicating that sensory organs on the
stinger are involved in identifying the cockroach’s
SupEG during the stinging process. The same treatment did not affect the duration of the thoracic
sting, as no neuronal tissue was removed from the
cockroach’s thorax.
What sensory modality does the wasp use to identify the cockroach’s SupEG? Possible candidates include mechanical, chemical, electrical, or behavioral
cues associated with the presence of the SupEG
inside the cockroach’s head capsule. When the
SupEG of cockroaches was replaced with a small
pellet of agarose prior to a wasp’s sting, the duration
of stinging in normal wasps depended on the concentration (density) of the agarose (Gal et al. 2014);
replacing the ganglion with a softer agarose pellet
induced a significantly prolonged head-sting, whereas
a harder agarose pellet induced a normal head-sting
(Fig. 2C). In addition, a pH indicator mixed with
these agarose pellets indicated that venom was injected into harder but not into softer pellets, suggesting that softer but not harder agarose pellets
triggered the wasp’s sensory feedback necessary for
injection of venom. These results demonstrate that
mechanical cues are at least sufficient to induce a
successful head-sting, whereas other experimental
manipulations ruled out electrical, chemical, or behavioral cues (Gal et al. 2014). Indeed, the wasp’s
stinger possesses several sensory organs (Fig. 2B),
among which are campaniform sensilla that are
known to work as mechanoreceptive strain gages
measuring bending of the cuticle, which may mediate
this process. Moreover, in vitro stimulation of the tip
of the stinger with an agarose pellet increased the
firing rate of afferent neurons ascending from the
stinger to the wasp’s ventral nerve cord in a
density-dependent manner, further strengthening
the hypothesis that the wasp uses mechanoreceptors
to differentiate the head ganglia from other tissues
within the cockroach’s head capsule (Gal et al. 2014).
Interestingly, the stinger also possesses contactchemoreceptive ‘‘dome-shaped sensilla;’’ however,
the function of these sensilla is yet unknown. It is
possible, for example, that these sensilla monitor the
amount of injected venom or mediate the host’s acceptance, as was previously demonstrated in other
parasitoid wasps (e.g., Nettles et al. 1982; Benedet
et al. 2002).
5
6
F. Libersat and R. Gal
30 min after the sting (Fouad et al. 1994), during
which the wasp searches for a suitable burrow to
conceal the host (Fig. 1). Grooming is known to
depress locomotion and increase the escape threshold of cockroaches (Camhi and Nolen 1981;
Hoganwarburg et al. 1995) and thus the venominduced excessive grooming behavior, which lasts
30 min, may act to keep the cockroach in the stinging site and enable the wasp to easily locate its prey
before transporting it to the nest. A similar phenomenon can be observed in cone snails, which use a
combination of venom components that induce hyperactivity followed by flaccid paralysis (Olivera
1999). Apparently, this uncoordinated and frantic
motor excitation immediately immobilizes the prey,
such that it cannot escape its predator until the
slower acting paralysis begins. The wasp may thus
use a similar strategy.
Another possibility is that the excessive grooming
behavior acts to remove ectoparasitic and fungal
infections from the cockroach’s cuticle, which may
be harmful for the wasp’s egg or its developing larva
(Nielsen et al. 2010; Zhukovskaya et al. 2013).
Recently, using behavioral observations and chemical
analyses, Herzner et al. (2013) showed that once the
larva penetrates into the cockroach’s abdomen, it
impregnates the cockroach from inside with large
amounts of an oral secretion containing a blend of
antimicrobials. This presumably provides an effective
frontline defense against the unpredictable spectrum
of pathogens with which the larva may be confronted
during its development inside the cockroach host.
The excessive grooming behavior may thus serve a
similar purpose in the pre-penetration stage, wherein
the egg and hatching larva are most vulnerable to
cockroach-borne ectoparasites and pathogens.
Mechanisms underlying the
venom-induced hypokinesia
Be the ultimate function of the excessive grooming
behavior as it may, this behavior decays in the stung
cockroach some 30 min following the head-sting. It is
at around this time that the second and more prominent effect of the head-sting commences, wherein
the cockroach ceases to initiate normal escape responses and spontaneous locomotion (Fouad et al.
1994; Libersat 2003). As the stung cockroach does
not seem to have lost the ability to walk per se,
this hypokinetic phase of envenomation has been
metaphorically described as a ‘‘lethargic,’’ ‘‘depressive-like’’ or even ‘‘zombie-like’’ state wherein the
cockroach appears to have lost its ‘‘will’’ or drive
to self-initiate locomotion (Gal and Libersat
2010a). Indeed, considering the extremely welladapted escape system of the American cockroach
(Nye and Ritzmann 1992; Schaefer et al. 1994),
such specific non-paralytic manipulation of the cockroach behavior in favor of the wasp’s needs appears
peculiar. It is interesting to note, however, that this
unique state is, in principle, reversible. Although in
nature the wasp’s larva kills the cockroach and
begins pupating in its abdomen 6 days after the
sting (Haspel et al. 2005), under experimental conditions (i.e., when the wasp is not allowed to lay an
egg on the cockroach or when the egg or larva are
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
Fig. 3 The wasp induces grooming in its cockroach host through the dopaminergic system (A) A cockroach grooming its antenna and
foreleg (arrows in top and bottom images, respectively) following a wasp’s sting. (B) Duration of grooming was significantly increased
in cockroaches that received a full sting (head þ thorax) compared with cockroaches that received a thoracic sting only and had their
SEG punctured by an experimenter (‘‘sham-sting’’). Dopamine (DA) injected into the hemolymph of an un-stung cockroach similarly
increased duration of grooming. (C) When injected into the SupEG, a DA-receptor (DAR) agonist induced a dose-dependent increase
in duration of grooming, with a maximal response at 10-8M. (D) Compared with a saline injection, a DAR antagonist but not an
octopamine-receptor (OAR) antagonist, prevented venom-induced grooming when injected into the hemolymph prior to a wasp’s sting.
Bars and data points represent means SD, **P50.01, ***P50.001 (t-test/ANOVA). Panels A, B, and D adapted from Weisel-Eichler
et al. (1999).
7
Wasp ‘zombifies’ cockroach host
removed by an experimenter) the cockroach regains
normal behavioral activity between 3 and 7 days after
the sting (Fouad et al. 1994; Gal and Libersat 2008).
Sensory and motor aspects of the venom-induced
hypokinesia
Central aspects of the venom-induced hypokinesia
The Jewel Wasp decreases the drive of its cockroach
prey to initiate and maintain walking
The hypokinetic state is specific to walking-related
behaviors, but does the venom completely prevent
the initiation of walking or does it elevate the threshold stimulus required to elicit walking? A recent
study (Gal and Libersat 2008) provides answers to
some of these questions.
Periplaneta are terrestrial insects, and therefore
immersion in water provides a reliable and continuous stressful stimulus that typically produces a rigorous walking-like motor pattern (Cocatrezilgien and
Delcomyn 1990). Hence, Gal and Libersat (2008)
quantified the walking-like behavior of cockroaches
immersed in a water-filled container, in a setup similar to the Forced Swim Test paradigm that is traditionally used to evaluate depression-like phenotypes
in non-human mammals (McArthur and Borsini
2006). Although stung cockroaches readily initiated
normal and coordinated walking-like motor patterns
upon immersion (Fig. 4C), their duration of swimming was dramatically lower compared with
un-stung cockroaches (Fig. 4D). In fact, stung cockroaches tended to float passively on the water, occasionally moving their antennae about in an
exploratory manner. Hence, in addition to increasing
the threshold for the initiation of walking-related behaviors, the venom appears to also affect the ability
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
American cockroaches are known for their highly
effective escape responses (Camhi 1988). For instance, when a predator approaches the cockroach,
wind-sensitive hairs on the cerci (two appendages at
the end of the abdomen) detect changes in air pressure resulting from the approaching predator, rapidly
convey this information through giant interneurons
(GIs) to the thoracic and head ganglia, and induce a
fast and reliable escape response in the opposite direction (Camhi 1988). Similarly, through the large
descending mechanosensory interneurons (DMIs)
reaching from the antennae to the head-ganglia
and thoracic ganglia, a tactile stimulus to the antenna also induces a reliable and rapid escape response (Ye and Comer 1996). Both these stimuli,
however, consistently fail to evoke such an escape
response in a stung cockroach (Fouad et al. 1996).
As this behavioral manipulation occurs only after the
venom is injected into the cockroach’s head, it
cannot directly affect the escape response at the
level of the leg muscles or neuromuscular junctions.
Instead, it could result from the venom’s effect on
sensory, central, or premotor neurons. As a first step,
therefore, the venom’s effect on sensory and premotor systems was tested.
Studies of the sensory systems in stung cockroaches revealed that sensory cues reliably propagate
along sensory neurons and interneurons, e.g. the GIs
from the cerci or the DMIs from the antennae
(Fouad et al. 1994, 1996). Similarly, the muscles
and motor neurons are functional in stung cockroaches and are able to produce normal motor outputs (Fouad et al. 1994, 1996; Gal and Libersat
2008). For instance, the rigorous escape response
evoked in normal cockroaches by tactile or wind
stimuli are characterized by the rhythmic recruitment
of slow and fast leg depressor and extensor muscles
acting in coordination (Libersat et al. 1999; Libersat
2003). As these muscles are innervated by specific
sets of thoracic motor neurons, electromyographic
(EMG) recordings from these muscles, for example,
through electrodes implanted in the coxal depressor
muscles of behaving cockroaches (Fig. 4A), provide a
direct readout of both the slow and fast coxal depressors (Ds and Df, respectively) as well as of their
associated motor neurons (Pearson and Iles 1971).
Such EMG recordings in stung cockroaches reveal
that, although a wind stimulus to the cerci evokes
neuronal activity that propagates normally to the
CNS, it evokes only a short burst of Ds potentials
and no Df potentials (Fig. 4B). Behaviorally, this is
associated with a postural reflex (Ds activity) without
the typical successive running response that is otherwise normally associated with a rapid phasic Df activity (Libersat et al. 1999). Nevertheless, both Ds
and Df potentials can be rhythmically evoked in a
walking-like manner when stung cockroaches are immersed in water (Gal and Libersat 2008) (Fig. 4C)
and Df potentials can be evoked normally when suspending stung cockroaches in the air and initiating
flying behavior, which involves Df activity (Libersat
2003). Hence, the venom must affect central, rather
than peripheral, mechanisms to decrease locomotion
and induce the ‘‘zombie-like’’ state. In addition,
these results imply that the venom does not affect
all motor outputs similarly but rather that some aspects of behavior, namely the initiation and/or maintenance of walking-related behaviors, are specifically
impaired in stung cockroaches while other motor
outputs are spared.
8
F. Libersat and R. Gal
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
Fig. 4 The motor behavior of cockroaches during the ‘‘zombie-like’’ state induced by the wasp’s sting (A) A diagram indicating
positioning of EMG electrodes (black circles). Numbers represent leg pairs (1, 2, and 3 for the prothoracic, mesothroacic, and
metathoracic legs, respectively) and letters represent lateralization (L: left, R: right). (B) EMG recordings from leg R3 in one control and
one stung cockroach standing on a solid surface. A wind stimulus applied to the cerci (bottom line in each trace) evoked rhythmic fast
and slow potentials (Df and Ds, respectively) in the depressor muscle of the coxa of a control cockroach, but only a burst of Ds
potentials (associated with a postural change without displacement) in a stung cockroach. Voltage scale is different for the top and
bottom traces. Adapted from Libersat (2003). (C) EMG recordings from legs R2, L3, and R3, and respective phase histograms, in one
control and one stung cockroach immersed in water. In both the control and the stung cockroach, a normal interleg coordination can
be observed during active bouts of swimming, such that legs R2 and L3 move in together (black bars in histograms) and alternate with
leg R3 (gray bars in histogram). Adapted from Gal and Libersat (2008). (D) Swimming trajectory (left) of one control and one stung
cockroach, and cumulative duration of swimming in a modified 60-s ‘‘forced swim test’’. Arrows and asterisks indicate location at the
beginning and end of the test, respectively. Bold lines indicate the spatial location during which periods of immobility were observed.
Bars represent means SD, ***P50.001 (t-test). Adapted from Gal and Libersat (2008).
9
Wasp ‘zombifies’ cockroach host
The Jewel Wasp decreases ongoing and evoked
neuronal activity in the cockroach’s SEG
The Jewel Wasp inserts its stinger through the cockroach’s soft neck cuticle in close proximity to the
location of the SEG, and venom is found in large
quantities specifically inside the SEG and SupEG of
stung cockroaches (Haspel et al. 2003). Both these
head ganglia are generally known to exert a ‘‘top–
down’’ regulation of the thoracic Central pattern
generators (CPGs), which in turn produce the local
spatiotemporal patterns required for locomotion
(Altman and Kien 1987; Strausfeld 1999; Strauss
2002; Gal and Libersat 2006). It is thus probable
that neuronal ensembles within either one, or both,
of these ganglia determine the ‘‘internal state’’ of the
cockroach, and thus its ‘‘motivation’’ to engage in
specific motor patterns such as walking.
Compared with un-stung cockroaches, spontaneous and stimulus-evoked neuronal spiking activity in
the core of the SEG was found to be dramatically
decreased in stung cockroaches (Gal and Libersat
2010b) (Fig. 5A and B), concomitant with the decrease in spontaneous and stimulus-evoked locomotion. This observation suggests that the SEG may be
an important part of the decision-making system and
may therefore play a central role in determining the
‘‘rest state’’ and the drive for walking in cockroaches.
Indeed, in cockroaches, the SEG is known generally
to exert a tonic permissive effect on walking-related
thoracic CPGs (Gal and Libersat 2006). Moreover,
intracellular recordings from neurons in the SEG
and SupEG of locusts indicate that the spontaneous
initiation of walking is accompanied by changes in
the firing pattern of several neurons descending from
both the SEG and SupEG. However, while SEG and
SupEG interneurons both fire during walking, and
are thus both involved in maintaining walking behavior, predominantly SEG interneurons fire during
the preparatory phase of walking. These observations
suggest a prime role for SEG neuronal circuits in
determining the motivational level of insects to
engage in walking (Kien and Altman 1992). Indeed,
some evidence also suggests that a venom-induced
decrease in SEG neuronal activity may be involved
in the ‘‘apathetic’’ state induced in crickets by the
parasitoid wasp Liris niger (Ferber et al. 2001).
The exact role of the SupEG in the venominduced manipulation of the cockroach motor
behavior is currently under investigation. Several
possibilities come to mind, for example, a role in
evoking the excessive grooming behavior (Libersat
2003) or a role in the venom-induced changes in
cockroach metabolism (Haspel et al. 2005). Because
the SupEG is known to regulate motor behavior in
insects (Roeder 1937; Ridgel and Ritzmann 2005; Gal
and Libersat 2006), it is also possible that the SupEG,
in concert with the SEG, plays a role in inducing
certain aspects of venom-induced hypokinesia. For
instance, the venom could affect the SupEG directly
by affecting specific circuitries in this ganglion, or
indirectly by affecting ascending SEG interneurons
which, in turn, modulate SupEG circuitries that control motor behavior. Several recent studies highlight
the role of a specific region within the SupEG,
namely the CC, in mediating numerous aspects of
walking behavior (Bender et al. 2010; Harley and
Ritzmann 2010; Guo and Ritzmann 2013). In fact,
the CC of arthropods shares numerous homologies
with the basal ganglia of vertebrates, implicating its
role in the regulation and release of adaptive behaviors (Strausfeld and Hirth 2013). Most of the venom
found in the SupEG of stung cockroaches is, indeed,
concentrated in and around the CC (Haspel et al.
2003).
Involvement of neuromodulatory systems in
venom-induced hypokinesia
Prime candidates for the hypokinetic manipulation
are neuromodulatory interneurons, and in particular
monoaminergic interneurons ascending from the
SEG to the SupEG. With respect to motivation, the
role of monoaminergic systems may have been conserved throughout evolution. In the CNS of insects
and mammals, DA and noradrenaline (or its invertebrate analog, OA) have been shown to profoundly
affect motivation, arousal, and locomotion (Libersat
and Pflueger 2004). Moreover, in insects as in mammals, it is the interplay between different neuromodulators that seems to affect behavioral choices.
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
of the cockroach to maintain such behaviors over a
long period of time. This, in turn, suggests that stung
cockroaches have a specific deficit in reaching the
decision to walk, or in their ‘‘drive’’ to initiate and
maintain walking, rather than in their ability to walk
per se. Understanding the mechanism underlying
such a manipulation may be beneficial in the investigation of key questions in the study of the neurobiology of behavior (Gal and Libersat 2010a), such as
which circuits determine the internal state of the
insect and regulate the decision to engage in a specific motor behavior, and what are the neuronal substrates responsible for determining the ‘‘motivation’’
specifically for walking? Although these questions are
at the core of current ongoing research, some answers have already begun to emerge owing to the
study of this unique parasitoid–host system.
10
F. Libersat and R. Gal
In the SEG, one specific population of monoaminergic neurons that may be affected by the venom is
the octopaminergic unpaired median neurons. The
axons of some of these neurons innervate segmental
ganglia while others innervate major neuropiles in
the SupEG (Braunig and Burrows 2004). Similar to
noradrenaline in mammals, OA in insects has been
previously implicated in regulating locomotion
(Sombati and Hoyle 1984; Bacon et al. 1995;
Pfluger and Duch 2000; Saraswati et al. 2004) and
activity in SEG-OA neurons in Manduca larvae has
been correlated with fictive locomotion (Cholewa
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
Fig. 5 Possible targets of the wasp’s venom (A) Representative traces of extracellular neuronal activity in the SEG of one control and
one stung cockroach. Adapted from Gal and Libersat (2010b). (B) Spontaneous neuronal activity in the SEG of stung cockroaches
is significantly lower than in control cockroaches. Bars represent means SEM, **P50.05 (t-test). Adapted from Gal and Libersat
(2010b). (C) Injection of an octopamine receptor (OAR) agonist into the SupEG increased spontaneous walking both in un-stung and
stung cockroaches. Adapted from Rosenberg et al. (2007). Bars represent means SD, ***P50.001 (ANOVA). (D) The foot-shock
voltage required to elicit an escape response, namely a ‘‘startle’’ response followed by locomotion, was reversibly increased following
the sting. Bars represent means SD, different letters represent statistically significant differences (P50.001, Mann–Whitney Rank Sum
Test). Adapted from Gal and Libersat (2008). (E) The ‘‘startle’’ threshold was similarly increased in stung cockroaches and in un-stung
cockroaches treated with an opioid receptor (OpR) agonist. Bars represent means SD. Adapted from Gavra and Libersat (2011).
11
Wasp ‘zombifies’ cockroach host
escaping the electric foot-shock (Gal and Libersat
2008) (Fig. 5D). This indicates that not only is the
threshold for initiation of walking elevated in stung
cockroaches, but also that their basic ability to recruit walking-related CPGs in a coordinated manner
upon reaching this threshold is preserved.
In search for a candidate neuromodulator to account for this change in the threshold for initiation
of startle, Gavra and Libersat (2011) showed that
injections of the opioid agonist loperamide induce
a significant and dose-dependent increase in the
avoidance threshold of cockroaches in a shuttle box
assay (Fig. 5E). Moreover, in the cockroach ‘‘antenna-heart’’ preparation [a well-known preparation
for testing the pharmacology of peptides in insects,
and which contains opioid fibers in cockroaches
(Gavra and Libersat 2011)], application of crude
venom completely inhibited contractions of the myogenic dilator muscle (Gavra and Libersat 2011).
Application of the opioid antagonist naloxone
(which, by itself, has no effect on muscle contractions) to the preparation partly reversed this effect by
normalizing the amplitude of the contractions but
not their frequency. Whether an opiate agonist in
the venom is directly responsible for the change in
escape threshold to noxious stimulus remains to be
proven.
Conclusions and future directions
In this article we unfold the mechanisms by which a
parasitoid wasp ‘‘hijacks’’ the CNS of its cockroach
host to control the host’s behavior. In a process involving mechanosensory inputs arriving from its
stinger, the Jewel Wasp identifies the cockroach’s
brain inside the head capsule, penetrates through
its ganglionic sheath (the insect’s blood–brain–
barrier) and injects a venom cocktail directly inside
the two ganglia of the head. This ‘‘brain-sting’’ modulates specific motor behaviors in the host, which
first grooms excessively and then shows a dramatic
decrease in the drive to self-initiate locomotion.
Unlike motivational deficits in mammals, however,
the venom-induced hypokinesia can be traced to specific neuronal structures within the CNS of stung
cockroaches; namely, it seems that the venom modulates neuronal circuitries within (at least) the cockroach’s SEG and/or CC in the SupEG, which may
therefore be involved in determining the ‘‘motivation’’ of the insect to engage in spontaneous and
evoked walking.
Although some candidates have been suggested,
the specific neuronal ensembles that are manipulated
by the venom have not been identified yet. Given the
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
and Pfluger 2009), further highlighting these neurons
as major candidates for the venom-induced hypokinesia. Accordingly, the OA receptor agonist chlordimeform has been shown to induce a significant
increase in spontaneous walking when injected into
the SupEG of either stung or un-stung cockroaches
(Rosenberg et al. 2007) (Fig. 5C). This correlates well
with the decrease in spontaneous neuronal spiking
activity in the core of the SEG in stung cockroaches
(as discussed above; Gal and Libersat 2010b), as OA
neurons are characterized by a baseline rate of firing
(Rosenberg et al. 2006). Thus, the Jewel Wasp’s
venom may interfere with octopaminergic modulation of the initiation of walking in central structures
of the cockroach SupEG, which receive inputs from
the SEG. A similar role for OA in depressing host’s
locomotion following a wasp’s sting has been also
proposed for the wasp Cotesia, a parasitoid of
Manduca (Adamo and Shoemaker 2000). Notably,
numerous studies have shown a dense innervation
of OA-immunoreactive neurons within the CC of
insects (Sinakevitch et al. 2005, 2011; Busch et al.
2009; Homberg et al. 2013); as mentioned above,
most venom injected by the Jewel Wasp inside the
cockroach’s SupEG is found in and around the CC
(Haspel et al. 2003).
Another possible target of the wasp’s venom is the
opioid system, which is known to affect responsiveness to stimuli not only in mammals but also in
insects (Ford et al. 1986; Gritsai et al. 2004).
Similar to the effect of the Jewel Wasp’s venom,
opioid-like substances are known to have an ‘‘analgesic’’-like effect in insects and therefore modulate
the insects’ threshold for escape (Stefano et al.
1990). Further, in fruit flies, enkephalin-like immunoreactive neurons are found in the CC and SEG
(Thorpe and Duve 1990), both implicated in the
control of walking in insects (Altman and Kien
1987; Strausfeld 1999; Strauss 2002; Gal and
Libersat 2006). To test the threshold for the initiation of startle in stung cockroaches, a modified
shuttle box was used in which escapable foot
shocks were administered to cockroaches’ legs without previous training. The voltage required to reliably elicit an escape response, which consists of a
‘‘startle’’ (‘‘nociceptive’’) response followed by a rigorous and rhythmic recruitment of the motor neurons and muscles associated with walking in thoracic
ganglion circuits, was noted at different time-points
after the sting. Compared with sham-treated cockroaches, this threshold voltage gradually increased
in stung cockroaches such that, at the peak of the
venom’s effect (2–4 h after the sting), stung cockroaches endured voltages 48-fold higher before
12
Acknowledgments
All experiments presented in this review comply with
Principles of Animal Care, NIH publication no. 86-23,
revised in 1985, and also with the current laws of the
State of Israel. We thank Gal Haspel for his wonderful illustrations of the venom’s behavioral effects, and
Dieter Schulten from the Aquazoo Löbbecke
Museum (Düsseldorf, Germany) for his kind gift of
wasps.
Funding
This work is currently supported by The Israel
Academy of Sciences and Humanities (1181/11).
References
Adamo SA, Shoemaker KL. 2000. Effects of parasitism on the
octopamine content of the central nervous system of
Manduca sexta: a possible mechanism underlying host
behavioural change. Can J Zool 78:1580–7.
Altman J, Kien J. 1987. Functional organization of the subesophageal ganglion in arthropods. In: Gupta AP, editor.
Arthropod brain; its evolution, development, structure, and
functions. New York: Wiley. p. 265–301.
Bacon JP, Thompson KS, Stern M. 1995. Identified octopaminergic neurons provide an arousal mechanism in the
locust brain. J Neurophysiol 74:2739–43.
Bender JA, Pollack AJ, Ritzmann RE. 2010. Neural activity in
the central complex of the insect brain is linked to locomotor changes. Curr Biol 20:921–6.
Benedet F, Leroy T, Gauthier N, Thibaudeau C, Thibout E,
Renault S. 2002. Gustatory sensilla sensitive to protein
kairomones trigger host acceptance by an endoparasitoid.
P Roy Soc B-Biol Sci 269:1879–86.
Braunig P, Burrows M. 2004. Projection patterns of posterior
dorsal unpaired median neurons of the locust subesophageal ganglion. J Comp Neurol 478:164–75.
Busch S, Selcho M, Ito K, Tanimoto H. 2009. A map of
octopaminergic neurons in the Drosophila brain. J Comp
Neurol 513:643–67.
Camhi JM. 1988. Escape behavior in the cockroach - distributed neural processing. Experientia 44:401–8.
Camhi JM, Nolen TG. 1981. Properties of the escape system
of cockroaches during walking. J Comp Physiol 142:339–46.
Cholewa J, Pfluger HJ. 2009. Descending unpaired median
neurons with bilaterally symmetrical axons in the suboesophageal ganglion of Manduca sexta larvae. Zoology
112:251–62.
Cocatrezilgien JH, Delcomyn F. 1990. Fast axon activity and
the motor pattern in cockroach legs during swimming.
Physiol Entomol 15:385–92.
Eberhard WG. 2000. Spider manipulation by a wasp larva.
Nature 406:255–6.
Ferber M, Horner M, Cepok S, Gnatzy W. 2001. Digger wasp
versus cricket: mechanisms underlying the total paralysis
caused by the predator’s venom. J Neurobiol 47:207–22.
Ford R, Jackson DM, Tetrault L, Torres JC, Assanah P,
Harper J, Leung MK, Stefano GB. 1986. A behavioral role
for enkephalins in regulating locomotor-activity in the
insect Leucophaea-Maderae - evidence for high-affinity
kappa-like opioid binding-sites. Comp Biochem Phys C
85:61–6.
Fouad K, Libersat F, Rathmayer W. 1994. The venom of the
cockroach-hunting wasp Ampulex-Compressa changes
motor thresholds - a novel tool for studying the neural
control of arousal. Zoolog Anal Complex Syst 98:23–34.
Fouad K, Libersat F, Rathmayer W. 1996. Neuromodulation
of the escape behavior of the cockroach Periplaneta americana by the venom of the parasitic wasp Ampulex compressa. J Comp Physiol A-Sens Neural Behav Physiol
178:91–100.
Gal R, Libersat F. 2006. New vistas on the initiation and
maintenance of insect motor behaviors revealed by specific
lesions of the head ganglia. J Comp Physiol A 192:1003–20.
Gal R, Libersat F. 2008. A parasitoid wasp manipulates the
drive for walking of its cockroach prey. Curr Biol
18:877–82.
Gal R, Libersat F. 2010a. On predatory wasps and zombie
cockroaches: Investigations of ‘‘free will’’ and spontaneous
behavior in insects. Commun Integr Biol 3:458–61.
Gal R, Libersat F. 2010b. A wasp manipulates neuronal activity in the sub-esophageal ganglion to decrease the drive for
walking in its cockroach prey. PLoS One 5:e10019.
Gal R, Kaiser M, Haspel G, Libersat F. 2014. Sensory arsenal
on the stinger of the parasitoid jewel wasp and its possible
role in identifying cockroach brains. PLoS One 9: e89683.
Gavra T, Libersat F. 2011. Involvement of the opioid system
in the hypokinetic state induced in cockroaches by a parasitoid wasp. J Comp Physiol A Neuroethol Sens Neural
Behav Physiol 197:279–91.
Gritsai OB, Dubynin VA, Pilipenko VE, Petrov OP. 2004.
Effects of peptide and non-peptide opioids on protective
reaction of the cockroach Periplaneta americana in the ‘‘hot
camera’’. J Evol Biochem Phys 40:153–60.
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
rich content of chemical compounds in the venom
(including monoamines, amino acids, peptides, and
proteins) it appears plausible that several neuromodulatory systems, such as the octopaminergic and
enkephalinergic ones, are targeted. Post-genomic
approaches may be used to answer this question, as
advances in this field have substantially increased our
understanding of the proximate mechanisms mediating hosts’ behavioral changes. For instance, hosts’
brain proteins can be extracted and their mass and
amino acid sequence determined via mass-spectrophotometry and aligned against available databases.
Given the time scale of the cockroach’s manipulation
by the Jewel Wasp, the venom may have genomic
effects on the cockroach’s CNS. Proteomics techniques can thus help identify the molecular targets
of different venom components within the cockroach’s brain. These lines of investigation hopefully
will provide useful information regarding the neuronal underpinnings of behavioral spontaneity, which
is a prime issue in behavioral neurobiology.
F. Libersat and R. Gal
Wasp ‘zombifies’ cockroach host
Moore EL, Haspel G, Libersat F, Adams ME. 2006. Parasitoid
wasp sting: a cocktail of GABA, taurine, and beta-alanine
opens chloride channels for central synaptic block and transient paralysis of a cockroach host. J Neurobiol 66:811–20.
Nettles WC, Morrison RK, Xie ZN, Ball D, Shenkir CA,
Vinson SB. 1982. Synergistic action of KCl and
magnesium-sulfate on parasitoid wasp oviposition. Science
218:164–6.
Nielsen C, Agrawal AA, Hajek AE. 2010. Ants defend aphids
against lethal disease. Biology Lett 6:205–8.
Nye SW, Ritzmann RE. 1992. Motion analysis of leg joints
associated with escape turns of the cockroach, Periplaneta
americana. J Comp Physiol A Sens Neural Behav Physiol
171:183–94.
O’Neill KM. 2001. Solitary wasps: behavior and natural history. Ithaca, N.Y: Comstock Pub. Associates.
Olivera BM. 1999. Conus venom peptides: correlating chemistry and behavior. J Comp Physiol A Sens Neural Behav
Physiol 185:353–9.
Pearson KG, Iles JF. 1971. Innervation of coxal depressor
muscles in the cockroach, Periplaneta americana. J Exp
Biol 54:215–32.
Pfluger HJ, Duch C. 2000. The functional role of octopaminergic neurons in insect motor behavior. Acta Biol Hung
51:343–8.
Quicke DLJ. 1997. Parasitic wasps. 1st edition. London, New
York: Chapman & Hall.
Ridgel AL, Ritzmann RE. 2005. Effects of neck and circumoesophageal connective lesions on posture and locomotion in
the cockroach. J Comp Physiol A Neuroethol, Sens Neural
Behav Physiol 191:559–73.
Roeder KD. 1937. The control of tonus and locomotor activity in the praying mantis (Mantis religiosa L.). J Exp Zool
76:353–74.
Rosenberg LA, Glusman JG, Libersat F. 2007. Octopamine
partially restores walking in hypokinetic cockroaches
stung by the parasitoid wasp Ampulex compressa. J Exp
Biol 210:4411–7.
Rosenberg LA, Pfluger HJ, Wegener G, Libersat F. 2006. Wasp
venom injected into the prey’s brain modulates thoracic
identified monoaminergic neurons. J Neurobiol 66:155–68.
Saraswati S, Fox LE, Soll DR, Wu CF. 2004. Tyramine and
octopamine have opposite effects on the locomotion of
Drosophila larvae. J Neurobiol 58:425–41.
Schaefer PL, Kondagunta GV, Ritzmann RE. 1994. Motion
analysis of escape movements evoked by tactile stimulation
in the cockroach Periplaneta americana. J Exp Biol
190:287–94.
Sinakevitch I, Mustard JA, Smith BH. 2011. Distribution of
the octopamine receptor AmOA1 in the honey bee brain.
PLoS One 6:e14536.
Sinakevitch I, Niwa M, Strausfeld NJ. 2005. Octopamine-like
immunoreactivity in the honey bee and cockroach: comparable organization in the brain and subesophageal ganglion.
J Comp Neurol 488:233–54.
Sombati S, Hoyle G. 1984. Central nervous sensitization and
dishabituation of reflex action in an insect by the neuromodulator octopamine. J Neurobiol 15:455–80.
Stefano G, Scharrer B, Lung M. 1990. Neurobiology
of opioids in Leucophaea maderae. In: Huber I,
Masler EP, Rao BR, editors. Cockroaches as models for
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
Grosman AH, Janssen A, de Brito EF, Cordeiro EG, Colares F,
Fonseca JO, Lima ER, Pallini A, Sabelis MW. 2008.
Parasitoid increases survival of its pupae by inducing
hosts to fight predators. PLos One 3:e2276.
Guo PY, Ritzmann RE. 2013. Neural activity in the central
complex of the cockroach brain is linked to turning behaviors. J Exp Biol 216:992–1002.
Harley CM, Ritzmann RE. 2010. Electrolytic lesions within
central complex neuropils of the cockroach brain affect
negotiation of barriers. J Exp Biol 213:2851–64.
Haspel G, Gefen E, Ar A, Glusman JG, Libersat F. 2005.
Parasitoid wasp affects metabolism of cockroach host to favor food preservation for its offspring. J Comp Physiol A
191:529–34.
Haspel G, Rosenberg LA, Libersat F. 2003. Direct injection
of venom by a predatory wasp into cockroach brain.
J Neurobiol 56:287–92.
Herzner G, Schlecht A, Dollhofer V, Parzefall C, Harrar K,
Kreuzer A, Pilsl L, Ruther J. 2013. Larvae of the parasitoid
wasp Ampulex compressa sanitize their host, the American
cockroach, with a blend of antimicrobials. Proc Natl Acad
Sci USA 110:1369–74.
Hoganwarburg AJ, Hogan JA, Ashton MC. 1995. Locomotion
and grooming in crickets - competition or time-sharing.
Anim Behav 49:531–3.
Homberg U, Seyfarth J, Binkle U, Monastirioti M,
Alkema MJ. 2013. Identification of distinct tyraminergic
and octopaminergic neurons innervating the central complex of the desert locust, Schistocerca gregaria. J Comp
Neurol 521:2025–41.
Kien J, Altman JS. 1992. Preparation and execution of movement: parallels between insect and mammalian motor systems. Comp Biochem Physiol Comp Physiol 103:15–24.
LeRalec A, Rabasse JM, Wajnberg E. 1996. Comparative morphology of the ovipositor of some parasitic hymenoptera
in relation to characteristics of their hosts. Can Entomol
128:413–33.
Libersat F. 2003. Wasp uses venom cocktail to manipulate
the behavior of its cockroach prey. J Comp Physiol A
Neuroethol Sens Neural Behav Physiol 189:497–508.
Libersat F, Delago A, Gal R. 2009. Manipulation of host
behavior by parasitic insects and insect parasites. Annu
Rev Entomol 54:189–207.
Libersat F, Gal R. 2007. Neuro-manipulation of hosts by parasitoid wasps. In: Rivers D, Yoder J, editors. Recent advances in the biochemistry, toxicity, and mode of action
of parasitic wasp venoms. Trivandrum: Research Signpost.
p. 93–114.
Libersat F, Haspel G, Casagrand J, Fouad K. 1999.
Localization of the site of effect of a wasp’s venom in the
cockroach escape circuitry. J Comp Physiol A Sens Neural
Behav Physiol 184:333–45.
Libersat F, Pflueger HJ. 2004. Monoamines and the orchestration of behavior. Bioscience 54:17–25.
McArthur R, Borsini F. 2006. Animal models of depression
in drug discovery: A historical perspective. Pharmacol
Biochem Be 84:436–52.
Molloy AG, Waddington JL. 1984. Dopaminergic behavior
stereospecifically promoted by the d1 agonist R-Sk-andF-38393 and selectively blocked by the D1 antagonist
Sch-23390. Psychopharmacology 82:409–10.
13
14
ovipositor apparatus and associated structures in the
Orussidae (Hymenoptera, Insecta). Zoomorphology
121:63–84.
Weisel-Eichler A, Libersat F. 2002. Are monoaminergic systems involved in the lethargy induced by a parasitoid wasp
in the cockroach prey? J Comp Physiol A 188:315–24.
Weisel-Eichler A, Haspel G, Libersat F. 1999. Venom of a
parasitoid wasp induces prolonged grooming in the cockroach. J Exp Biol 202(Pt 8):957–64.
Ye SP, Comer CM. 1996. Correspondence of escape-turning
behavior with activity of descending mechanosensory
interneurons in the cockroach, Periplaneta americana.
J Neurosci 16:5844–53.
Yellman C, Tao H, He B, Hirsh J. 1997. Conserved and sexually dimorphic behavioral responses to biogenic amines in
decapitated Drosophila. Proc Natl Acad Sci USA 94:4131–6.
Zhukovskaya M, Yanagawa A, Forschler BT. 2013. Grooming
behavior as a mechanism of insect disease defense. Insects
4:609–30.
Downloaded from http://icb.oxfordjournals.org/ at Ben Gurion University - Aranne Library on April 5, 2014
neurobiology: applications in biomedical research. Boca
Raton, FL: CRC Press. p. 285–302.
Strausfeld NJ. 1999. A brain region in insects that supervises
walking. Prog Brain Res 123:273–84.
Strausfeld NJ, Hirth F. 2013. Deep homology of arthropod
central complex and vertebrate basal ganglia. Science
340:157–61.
Strauss R. 2002. The central complex and the genetic dissection of locomotor behaviour. Curr Opin Neurobiol
12:633–8.
Thorpe A, Duve H. 1990. Morphological, biochemical, and
physiological-studies on insect enkephalins. Prog Clin Biol
Res 342:293–9.
Vilhelmsen L. 2000. The ovipositor apparatus of basal
Hymenoptera (Insecta): phylogenetic implications and
functional morphology. Zool Scr 29:319–45.
Vilhelmsen L, Isidoro N, Romani R, Basibuyuk HH,
Quicke DLJ. 2001. Host location and oviposition in a
basal group of parasitic wasps: the subgenual organ,
F. Libersat and R. Gal